Widespread gene delivery to motor neurons using peripheral injection of AAV vectors

ABSTRACT

The present invention relates to compositions and methods, in particular to methods based on systemic injection of rAAV, for delivering genes to cells of the central nervous system in mammals, such as brain neurons or glial cells, and in particular to motor neurons or glial cells of the spinal cord The invention also relates to methods of treating motor neuron disorders in mammals by expression of therapeutic genes. The invention stems from the unexpected discovery that peripheral injection of AAV vectors leads to a bypass of the blood brain barrier and a massive infection of motor neurons. The invention may be used in any mammal, including human subjects.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/713,347, filed Sep. 22, 2017, which is a divisional of U.S.application Ser. No. 12/734,016, filed May 28, 2010, now U.S. Pat. No.9,926,574, which is the U.S. Natl. Stage of PCT/EP2008/063297, filedOct. 3, 2008, which claims the benefit of European Application EP07301435.9, filed Oct. 5, 2007. All of these applications areincorporated by reference in their entirety.

The present invention relates to compositions and methods for deliveringgenes to cells of the central nervous system in mammals. The inventionalso relates to methods of treating motor neuron disorders in mammals byexpression of therapeutic genes. The invention stems from the unexpecteddiscovery that peripheral injection of AAV vectors leads to a bypass ofthe blood brain barrier and a massive infection of motor neurons, aswell as other cells in the central nervous system. The invention may beused in any mammal, including human subjects.

INTRODUCTION

Motor neuron (MN) diseases, such as spinal muscular atrophy (SMA),amyotrophic lateral sclerosis (ALS) or Kennedy's disease, areneurodegenerative disorders characterised by the selective degenerationof MNs in the spinal cord, brainstem and/or motor cortex (Monani 2005;Pasinelli and Brown 2006); (MacLean, Warne et al. 1996). There is notreatment for these diseases, mostly because drug delivery to MN viasystemic injections is hindered by the presence of the“blood-brain-barrier” (BBB). This anatomical and physiological barrieris formed by tight junctions between the endothelial cells of thecentral nervous system (CNS) capillaries and prevents easy passage ofmolecules between the circulation and the CNS (Scherrmann 2002). Thealternative supply of MN with recombinant proteins injected directlyinto the CNS parenchyma is also difficult due to the invasiveness of thesurgical procedure hampering a potential clinical application.

Failure of the classical pharmacology has led the scientific communityto develop new therapeutic strategies based, in particular, on genetransfer technology using viral vectors. However, conventional viralvectors generally do not pass the BBB, and the first proposed genetransfer strategies included intrathecal delivery or direct injectionsof the vectors into the spinal cord parenchyma (Davidson, PNAS 2000)(Azzouz, Hottinger et al. 2000). However, these invasive approachesfailed to produce efficient widespread CNS transduction. Injection ofthe viral vectors into the cerebral ventricles was also used in the aimto transduce the epithelial cells of the choroids plexus and ependyma,mediating secretion of the therapeutic proteins in the cerebrospinalfluid (CSF) and further diffusion through the CNS parenchyma (Passiniand Wolfe 2001). However, diffusion of the recombinant proteins to thewhole nervous tissue was far from being optimal, and again, thepotential risks related to the surgical procedure is an obstacle to theclinical application of this method.

An alternative non-invasive strategy was further developed usingretrograde axonal transport of viral vectors to MN through intramuscular(i.m.) injections. Gene vectors such as adenovirus, adeno-associatedvector (AAV) or equine-anemia viruses pseudotyped with the rabies Gglycoprotein (EIAV) indeed undergo retrograde transport along the MNaxons after i.m. injections, and were successfully used to transducelower MN in experimental animals (Finiels et al., 1995; Kaspar et al.,2003; Azzouz et al., 2004). However, the clinical value of this methodremains questionable due, in particular, to the large number of bothinjection sites and viral particles that would be needed for targetingMN in pathologies that affect most of the patient's motor units.

In order to counteract these difficulties, we tested the efficiency forMN transduction of new AAV serotypes and genomes after intramuscular(i.m.), intravenous (i.v.) and intraperitoneal (i.p.) delivery in mice.In particular, we compared the efficiency of recombinant single-strandedand self-complementary AAV vectors (ssAAV and scAAV, respectively) ofserotype 1 and 9 for mediating CNS transduction in mice.

Our main results demonstrate that recombinant AAV vectors (e.g., scAAV9)are particularly efficient to transduce spinal cord MNs after i.v.delivery in mice. Furthermore, we show the feasibility of this method ina large animal model, a domestic cat model of autosomal recessive SMAsimilar to human SMA type III, associated to deletions of the LIX1 gene(Fyfe et al, 2006). Our method also allows to transduce other cells ofthe CNS, including glial cells, neurons in the hippocampus and habenularnuclei, and astrocytes. This invention thus shows, for the first time,that it is possible to transfer genes of interest to MNs after a singlei.v. injection in mice, achieving broad gene delivery to the spinal cordand/or other nervous cells, therefore, offering new avenues for thetreatment of MN diseases.

SUMMARY OF THE INVENTION

The present invention relates to novel compositions and methods for thedelivery of therapeutic products to the CNS using recombinant AAVvectors. More specifically, the invention relates to compositions andmethods for delivering genes into the motor neurons or glial cells ofmammalian subjects through peripheral administration of AAV vectors.

An object of this invention more specifically relates to the use of anAAV vector comprising a gene of interest (e.g., encoding a therapeuticor diagnostic product) for the manufacture of a medicament fordelivering the gene to cells in the central nervous system, particularlymotor neurons or glial cells, by peripheral administration of said AAVvector to said subject.

An other object of this invention relates to the use of an AAV vectorcomprising a gene of interest (e.g., encoding a therapeutic ordiagnostic product) for the manufacture of a medicament for deliveringthe gene to spinal cord motor neurons by peripheral administration ofsaid AAV vector to said subject.

A further object of this invention resides in a method of delivering agene to cells in the central nervous system, particularly motor neuronsor glial cells, in a mammal, the method comprising administering to themammal by peripheral route an AAV vector comprising said gene, saidadministration allowing infection of cells in the central nervoussystem, particularly motor neurons or glial cells, by said AAV vectorsand thereby delivery of said gene to cells in the central nervoussystem, particularly motor neurons or glial cells.

An object of this invention also relates to the use of an AAV vectorcomprising a therapeutic gene for the manufacture of a medicament fortreating a motor neuron disorder in a subject, wherein said AAV vectoris administered by peripheral injection to said subject, saidadministration causing infection of (spinal cord) motor neurons andexpression of the gene in (spinal cord) motor neurons.

Another object of this invention relates to the use of an AAV vector forthe manufacture of a medicament for producing a therapeutic protein orRNA into (spinal cord) motor neurons of a subject by peripheralinjection of said vector.

The invention also relates to the use of an AAV vector for delivering agene to cells in the central nervous system, particularly motor neuronsor glial cells, by crossing the blood brain barrier.

The invention also relates to a method of gene therapy across the bloodbrain barrier in a mammalian subject, the method comprising theperipheral administration of an AAV vector to the subject.

A further object of this invention is a method of genetically modifyingcells in the central nervous system, particularly motor neurons in amammalian subject, the method comprising peripherally administering AAVvectors to the subject.

The invention also relates to the use of an AAV vector for themanufacture of a medicament for delivering a gene to the spinal cord byperipheral administration of the AAV vector.

The invention also resides in a method of gene delivery to the spinalcord of a subject, the method comprising peripherally administering tothe subject an AAV vector comprising said gene.

LEGEND TO THE FIGURES

FIG. 1 a-c . Widespread gene delivery to the neonatal mouse muscles andCNS after intramuscular AAV injection (blue: mSEAP histochemicalstaining). Representative cross sections of the (a) gastrocnemius muscle(b) brain (3rd ventricle) and (c) spinal cord at 3 days (PN4) or 7 days(PN8) after injection of ss- or scAAV1 or AAV9.

NI: non-injected; PN4, post-natal 4; PN8, post-natal 8; ss,single-strand; sc: self-complementary. Scale bar (b, c) 100 μm.

FIG. 2 a-d . Widespread gene delivery to the neonatal mouse muscles andCNS after intraperitoneal AAV injection (blue: mSEAP histochemicalstaining). Representative tissue sections of the (a) diaphragm muscle(b) cerebral 3^(rd) ventricle (arrows: mSEAP expressing choroids plexuscells; arrowheads: ependymal cells) (c) CNS parenchyma (arrows: neuronalcells).

NI: non-injected; PN4, post-natal 4; PN8, post-natal 8; ss,single-strand; sc: self-complementary. Scale bar (a, b, c) 100 μm; (d)40 μm.

FIG. 3 a-h . Intraperitoneal delivery of self-complementary AA9-GFPmediates CNS transduction in neonatal mice. Representative brain andspinal cord cross sections treated for GFP immunohistochemistry 7 daysafter AAV delivery. Transgene expression was detected in (a) thechoroids plexus epithelial cells (b) hippocampal cells with a neuronal(arrowhead and top box) and glial (arrow and bottom box) morphology (c)cells of the entorhinal cortex (arrows indicate cells with a typicalneuronal morphology) (d, e) cells of the spinal cord (the arrowindicates a GFP-labelled cell with a motor neuron morphology) and (f)sensory fibers in the cervical spinal cord. Scale bar 40 μm.

FIG. 4 a-f . Intramuscular delivery of self-complementary AAV9 vectorsallows transduction of CNS cells in neonatal mice. Brain and spinal cordhistological sections were treated with GFP immunohistochemistry 7 daysafter AAV injection. Transgene expression was detected in (a) theepithelial cells of the choroids plexus (arrow) and the ependyma(arrowheads) (b,c) neural cells of the septum and (d, e) of theentorhinal cortex and (f) the corticospinal tract at the level of thepyramidal decussation in the cervical spinal cord (arrows). Scale bar 20μm.

FIG. 5 a-m . Intravenous delivery of self-complementary AAV9 vectorsmediate GFP expression in the CNS of neonatal mice. Representativephotomicrographs of brain and spinal cord histological sections treatedfor GFP immunostaining 7 days after AAV injection. GFP-positive cellswere detected in (a) the epithelial cells of the choroids plexus (arrow)and the ependyma (arrowheads) (c) brain blood vessels (d,f) hippocampalcells with a neuronal (arrow) and glial (arrowhead) morphology (g)neuron-like cells of the entorhinal cortex. Many motor neuron-like cellbodies (arrows) and fibres (arrowheads) were efficiently transducedthroughout the spinal cord at the (h) cervical (i) thoracic and (j)lumbar levels. No staining was observed in the CNS of uninjected mice asshown in representative sections from (b) the 3rd ventricle or (e) thehippocampus or (k-m) the spinal cord. Scale bar (a, b) 100 μm, (c, d, f)40 μm, (e, g) 100 μm, (h-m) 20 μm.

FIG. 6 a-e . GFP-expressing single-stranded AAV9 vectors mediatetransgene expression in the CNS of neonatal mice. Representativephotomicrographs of brain and spinal cord sections from neonatal micetreated for GFP-immunohistochemistry 3 weeks after i.v. injection ofssAAV9 vectors. GFP-positive cells in (a) the choroids plexus(asterisk), the hippocampus (arrowhead and box) and the habenularnucleus (arrow) (b) the median eminence and (c-e) motor neuron-likecells in the ventral spinal cord. Scale bar panels b: 100 μm; c, d, e:20 μm.

FIG. 7 a-h . Recombinant AAV9 vectors mediate transgene expression inthe adult mouse CNS. Representative coronal brain sections from adultC57bl6 mice 4 weeks after intravenous delivery of 3×10¹¹ (b) or 1×10¹²(c-h) vector genome of mSEAP-expressing scAAV9 (a,c), ssAAV9 (b), scAAV1(d) and ssAAV1 (e); Scale bar panels g, h, j: 100 μm; panels i, k, l: 20μm.

FIG. 8 a-g . Intravenously injected recombinant AAV9 vectors mediatetransgene expression in the spinal cord of adult mice. Representativetransversal spinal cord sections from adult C57bl6 mice 4 weeks afterintravenous delivery of 1×10¹² vector genome of mSEAP-expressing ssAAV9(a,b), scAAV9 (c,g). Scale bars (a,b,e): 40 μm, boxes: 20 μm; (c,d): 100μm; (f): 50 μm; (g): 20 μm.

FIG. 9 a-d . Intravenous injection of AAV9-GFP in LIX-1 cats mediatestransgene expression throughout the spinal cord. Representativetransversal spinal cord sections from a 2 days-old LIX1 heterozygous catwere observed using laser scanning confocal microscopy (FIG. 9 a,c ) ortreated for GFP immunohistochemistry (FIG. 9 b,d ) 10 days afterinjection of GFP-expressing scAAV9 into the jugular vein.

Scale bars (a): 200 μm; (b, d): 50 μm; (c): 100 μm.

FIG. 10 a-f . Intravenous injection of GFP expressing AAV9 (1.5×10+12vector genome-containing particles of scAAV9-CMV-eGFP) in LIX-1 catsmediates transgene expression in motor neurons. A double-immunostaininganalysis using antibodies against GFP and choline acetyl transferase(ChAT) showed that, in both SMA-affected (a-c) and non-affected kitten(d-f), a significant part of the GFP-positive cells were motor neurons.

FIG. 11 a-l . Widespread spinal cord transduction is mediated by i.v.delivery of highly concentrated scAAV9 in adult mice.

High GFP expression in neuronal (arrows) and glial (arrowheads) cells in(a-c) cervical and (d,e) lumbar spinal cord (f) dorsal root ganglionsections treated for GFP-immunostaining 4 weeks after i.v. injection of2×10¹² vg scAAV9. (g-l) Double immunofluorescence analysis for (g, j)GFP and (h, k) GFAP (glial fibrillary acidic protein, a marker ofastrocytes, red) shows GFP expression in some astrocytes (arrowheadsindicate double-labeled cells). (i, l) Merge. Scale bars (a, b, d-l): 50μm, (c): 20 μm.

DETAILED DESCRIPTION OF THE INVENTION

Widespread gene delivery to the spinal cord is an important challengefor the treatment of motor neuron (MN) diseases such as spinal muscularatrophy (SMA) or amyotrophic lateral sclerosis (ALS). Here, we describea new gene transfer methodology that allows efficient MN transductionafter a single peripheral injection of recombinant AAV vectors. Weinjected recombinant single strand (ss) and self-complementary (sc) AAVvectors of serotype 1 and 9 intraperitonealy, intramuscularly orintravenously (i.v.) in neonatal or adult mice and analyzed transgeneexpression in the central nervous system (CNS). Both recombinant ss- andscAAV9 vectors were found to target neural and epithelial brain cellsand, importantly, motor neurons and glial cells in the spinal cord. Thedorsal sensory fibers and the dorsal root ganglions also appeared highlytransduced. The most impressive transduction efficacy was obtained withi.v. injected scAAV9 vectors. We further confirmed the ability of i.v.injected scAAV9 to bypass the blood-brain barrier and transduce lowermotor neurons in a feline model of SMA. This strategy represents thefirst non invasive procedure that achieves widespread transgene deliveryto the spinal cord, offering new avenues for the treatment of MNdiseases.

AAV Vectors

Within the context of the present invention, the term “AAV vector”designates any vector which comprises or derives from components of AAVand is suitable to infect mammalian cells, preferably human cells. Theterm AAV vector typically designates an AAV type viral particle (orvirion) comprising at least a nucleic acid molecule encoding atherapeutic protein. As will be discussed below, the AAV may be derivedfrom various serotypes, including combinations of serotypes (i.e.,“pseudotyped” AAV) or from various genomes (e.g. single-stranded orself-complementary). In addition, the AAV vector may be replicationdefective and/or targeted.

Adeno-associated virus (AAV) is a dependent parvovirus, of approximatelytwenty nanometers in size. Like other parvoviruses, AAV is asingle-stranded, non-enveloped DNA virus, having a genome of about 5000nucleotides in length, containing two open reading frames. The left-handopen reading frame codes for the proteins responsible for replication(Rep), while the right-hand open reading frame encodes the structuralproteins of the capsid (Cap). The open reading frames are flanked by twoITR sequences, which serve as the origin of replication of the viralgenome. Furthermore, the genome also contains a packaging sequence,allowing packaging of the viral genome into an AAV capsid.

AAV requires co-helper functions (which may be provided e.g. by anadenovirus, or by suitable packaging cells or helper plasmids) toundergo a productive infection in cultured cells. In the absence of suchhelper functions, the AAV virions essentially enter the cells, migrateto the nucleus as a single-stranded DNA molecule, and integrate into thecells' genome. AAV has a broad host range for infectivity, includinghuman cells, is ubiquitous in humans, and is completely non-pathogenic.

AAV vectors have been designed, produced and used to mediate genedelivery in human subjects, including for therapeutic purposes. Clinicaltrials are presently ongoing in various countries using AAV vectors.Typically, AAV vectors for use in gene transfer comprise a replicationdefective AAV genome lacking functional Rep and Cap coding viralsequences. Such replication defective AAV vectors more preferably lackmost or all of the Rep and Cap coding sequences, and essentially retainone or two AAV ITR sequences and a packaging sequence.

Methods of producing such AAV vectors have been disclosed in theliterature, including using packaging cells, auxiliary viruses orplasmids, and/or baculovirus systems (Samulski et al., (1989) J.Virology 63, 3822; Xiao et al., (1998) J. Virology 72, 2224; Inoue etal., (1998) J. Virol. 72, 7024; WO98/22607; WO2005/072364). Methods ofproducing pseudotyped AAV vectors have also been reported (e.g.,WO00/28004), as well as various modifications or formulations of AAVvectors, to reduce their immunogenicity upon in vivo administration (seee.g., WO01/23001; WO00/73316; WO04/112727; WO05/005610; WO99/06562).

AAV vectors may be prepared or derived from various serotypes of AAVs,which may be even mixed together or with other types of viruses toproduce chimeric (e.g. pseudotyped) AAV viruses.

In a particular embodiment, the AAV vector for use in the presentinvention is derived from a human AAV virus.

Such a human AAV (capsid and ITR) may be derived from any knownserotype, e.g. from any one of serotypes 1-11, preferably from AAV2,AAV4, AAV6, AAV8 and AAV9, more preferably from AAV6, AAV8 and AAV9,even more preferably from AAV9. Specific examples of such AAV vectorsare vectors comprising an AAV2-derived genome (a nucleic acid moleculecomprising an AAV2-derived ITR and an AAV2-derived packaging sequence,operatively linked to a nucleic acid encoding a therapeutic protein,preferably two AAV2-derived ITR flanking an AAV2-derived packagingsequence and a nucleic acid encoding a therapeutic protein) in anAAV2-derived capsid; vectors comprising an AAV4-derived genome in anAAV4-derived capsid; vectors comprising an AAV6-derived genome in anAAV6-derived capsid; vectors comprising an AAV8-derived genome in anAAV8-derived capsid; vectors comprising an AAV9-derived genome in anAAV9-derived capsid

In another particular embodiment, the AAV vector is a pseudotyped AAVvector, i.e. comprises sequences or components originating from at leasttwo distinct AAV serotypes. In a particular embodiment, the pseudotypedAAV vector comprises an AAV genome derived from one AAV serotype (forexample AAV2), and a capsid derived at least in part from a distinct AAVserotype. Specific examples of such pseudotyped AAV vectors include,without limitation, vectors comprising an AAV2-derived genome in anAAV4-derived capsid; or vectors comprising an AAV2-derived genome in anAAV6-derived capsid; or vectors comprising an AAV2-derived genome in anAAV8-derived capsid; or vectors comprising an AAV2-derived genome in anAAV9-derived capsid;

In a further particular embodiment, which may be combined with any ofthe above embodiments, the AAV vector may comprise a modified capsid,including proteins or peptides of non viral origin or structurallymodified, to alter the tropism of the vector. As a particular example,the capsid may include a ligand of a particular receptor, or a receptorof a particular ligand, to target the vector towards cell type(s)expressing said receptor or ligand, respectively.

In the AAV vectors used in the present invention, the AAV genome may beeither a single stranded nucleic acid or a double stranded, selfcomplementary nucleic acid (McCarty et al., Gene Therapy, 2001), morepreferably a self complementary nucleic acid.

As discussed above, the AAV-derived genome comprises a nucleic acidencoding a therapeutic protein. Typically, the nucleic acid alsocomprises regulatory sequences allowing expression and, preferably,secretion of the encoded protein, such as e.g., a promoter, enhancer,polyadenylation signal, internal ribosome entry sites (IRES), sequencesencoding protein transduction domains (PTD), and the like. In thisregard, the nucleic acid most preferably comprises a promoter region,operably linked to the coding sequence, to cause or improve expressionof the therapeutic protein in infected cells. Such a promoter may beubiquitous, tissue-specific, strong, weak, regulated, chimeric, etc., toallow efficient and suitable production of the protein in the infectedtissue. The promoter may be homologous to the encoded protein, orheterologous, including cellular, viral, fungal, plant or syntheticpromoters. Most preferred promoters for use in the present inventionshall be functional in nervous cells, particularly in human cells, morepreferably in motor neurons. Examples of such regulated promotersinclude, without limitation, Tet on/off element-containing promoters,rapamycin-inducible promoters and metallothionein promoters. Examples ofpromoters specific for the motor neurons include the promoter of theCalcitonin Gene-Related Peptide (CGRP), a known motor neuron-derivedfactor. Other promoters functional in motor neurons include thepromoters of Choline Acetyl Transferase (ChAT), Neuron Specific Enolase(NSE), Synapsin, or ubiquitous promoters including Neuron SpecificSilencer Elements (NRSE). Examples of ubiquitous promoters include viralpromoters, particularly the CMV promoter, the RSV promoter, the SV40promoter, etc. and cellular promoters such as the PGK (phosphoglyceratekinase) promoter.

In a preferred embodiment, the nucleic acid comprises a leader sequenceallowing secretion of the encoded protein. Fusion of the transgene ofinterest with a sequence encoding a secretion signal peptide (usuallylocated at the N-terminal of secreted polypeptides) will allow theproduction of the therapeutic protein in a form that can be secretedfrom the transduced cell into the CSF. Examples of such signal peptidesinclude the albumin, the 3-glucuronidase, the alkaline protease or thefibronectin secretory signal peptides.

According to another specific embodiment, the transgene is fused withPTD sequences, such as the Tat or VP22 sequences, in order to cause orimprove secretion of the therapeutic protein from the transduced cellsand re-uptake by neighbour ones.

In a particular embodiment the nucleic acid comprises, operably linker,a promoter and a leader sequence, to allow expression and secretion ofthe encoded protein.

In a further particular embodiment, the nucleic acid comprises, operablylinker, a promoter, a leader sequence and a PTD sequence, to allowexpression and secretion of the encoded protein.

In a most preferred embodiment, the promoter is specific or functionalin motor neurons, i.e., allows (preferential) expression of thetransgene in said cells.

As discussed above, the AAV vectors may be produced by techniques knownper se in the art, as further illustrated in the examples.

Peripheral Administration

The invention is based on the unexpected discovery that effective andwidespread expression of genes into motor neurons or glial cells can beachieved with non-invasive techniques, through peripheral administrationof AAV vectors. Such peripheral administration includes, withoutlimitation, any administration route which does not imply directinjection into the brain. More particularly, peripheral administrationcomprises systemic injections, such as intramuscular (i.m.), intravenous(i.v.), intraperitoneal (i.p.), intra-arterial, sub-cutaneous ortransdermic injections. Peripheral administration also includes oraladministration of AAV vectors (WO96/40954), delivery using implants(WO01/91803), or administration by instillation through the respiratorysystem, e.g., using sprays, aerosols or any other appropriateformulations.

Most preferred peripheral administration includes peripheral injection,in particular systemic injection, most preferably i.m., i.p. or i.v.injection.

The doses of AAV vectors may be easily adapted by the skilled artisan,e.g., depending on the disease condition, the subject, the treatmentschedule, etc.

Typically, from 10⁹ to 10¹⁴ viral genomes (transducing units) areadministered per dose in mice, preferably from about 10¹¹ to 10¹³.

Typically, the doses of AAV vectors to be administered in humans mayrange from 10¹¹ to 10¹⁷ viral genomes, preferably from 10¹³ to 10¹⁶.

A preferred effective dose within the context of this invention is adose allowing an optimal transduction of the spinal cord cells (motorneurons and/or glial cells).

The AAV vector may be administered in any suitable form, either as aliquid solution or suspension, as a solid form suitable for solution orsuspension in liquid prior to injection, as a gel or as an emulsion. TheAAV vectors are typically formulated with any appropriate andpharmaceutically acceptable excipient, carrier, adjuvant, diluent, etc.For injection, the excipient may be a liquid, isotonic solution, buffer,such as a sterile and pyrogen-free water or a sterile and pyrogen-freephosphate-buffered saline solution. For inhalation, the excipient may bein particulate form.

The AAV vectors are typically administered in a“therapeutically-effective” amount, i.e., an amount that is sufficientto alleviate (e.g., decrease, reduce) at least one of the symptomsassociated with the disease state, or to provide improvement in thecondition of the subject. It should be pointed out that repeatedadministrations may be performed, if required, using either the same ordifferent peripheral administration route and/or the same or distinctAAV serotypes.

The inventors have shown for the first time that AAV vectors, inparticular scAAV vectors, administered peripherally cross the bloodbrain barrier and cause substantial infection of CNS cells. This effectis obtained without the need of using blood-brain barrier disruptingagents. Hyperthermia, mannitol, bradykinin and NS1619 are illustrativeblood-brain barrier disrupting agents.

Accordingly, in a particular embodiment, the invention relates to an useor method as defined above, comprising peripheral administration of anAAV vector, preferably a scAAV vector, more preferably a scAAV9 vector,wherein no blood-brain barrier disrupting agent is implemented.Furthermore, the invention relates to an use or method as defined above,wherein no mannitol is injected to the subject.

Alternatively, in another particular embodiment, the invention relatesto an use or method as defined above, further comprising disruption ofthe blood-brain barrier with a blood-brain barrier disrupting agent orprocess, to further increase the crossing of the scAAV vectorsimplemented in the present invention through the blood-brain barrier.

Motor Neuron Disorder

The invention shows, for the first time, that AAV vectors administeredperipherally cross the blood brain barrier and cause substantialinfection of CNS cells, particularly of motor neurons throughout thespinal cord. The results presented show that infection is effective fromthe cervical segment to the lumbar segment of the spinal cord, therebyproviding a widespread gene delivery into motor neurons.

The invention may be used to treat a variety of disorders throughdelivery of a therapeutic product into CNS cells including the motorneurons. The therapeutic product may be any protein, peptide or RNA thatmay alleviate or reduce symptoms that result from an absence or defectin a protein in a cell or subject or that otherwise confers a benefit toa subject. Examples of therapeutic proteins include growth factors,cytokines, hormones, neurotransmitters, enzymes, anti-apoptotic factors,angiogenic factors, and any protein known to be mutated in pathologicaldisorders such as the “survival of motor neuron” protein (SMN). Examplesof therapeutic RNA include antisense RNA or RNAi targeting messengerRNAs coding for proteins having a therapeutic interest in any of thediseases mentioned herein below. For example, an RNAi targeting thesuperoxide dismutase enzyme may be coded by an AAV vector as definedabove, in view of the treatment of ALS.

Depending on the therapeutic product, the invention can be used to treatvarious diseases, including any disease which may be treated orprevented by expressing therapeutic proteins into nervous tissue. Suchdiseases include CNS disorders, preferably selected fromneurodegenerative diseases, neuromuscular diseases, lysosomal diseases,trauma, bone marrow injuries, pain (including neuropathic pain), cancersof the nervous system, demyelinating diseases, autoimmune diseases ofthe nervous system, neurotoxic syndromes, sleeping disorders.

Specific examples of diseases include Alzheimer's Disease, Parkinson'sDisease, Huntington's Disease, Tourette Syndrome, schizophrenia, Slydisease, Hunter's disease, dementia, paranoia, obsessive compulsivedisorder, learning disabilities, amyotrophic lateral sclerosis, spinalmuscular atrophy, Charcot-Marie Tooth disease, spinocerebellar ataxia,spastic paraplegia, Kennedy's disease, glioblastoma, neuroblastoma,autism, Gaucher's disease, Hurler's disease, Krabbe's disease andaltered behaviors (e. g., disorders in sleeping, perception orcognition).

The invention may be used in any mammalian, particularly in humansubjects, including adults, for preventive or curative treatment.

The invention can also be used in diagnostic methods, to detect thestatus or activity or growth of motor neurons in mammalian subjects. Forsuch indications, the vector typically comprises a detectable gene(fluorescent, luminescent, etc.) and is used as a marker.

The invention can also be used in animal subjects, e.g., to assist inthe research of candidate drugs for treating CNS disorders and/or tounderstand the mechanisms of motor neuron growth, differentiation,activity, etc.

Further aspects and advantages of the present inventions will bedisclosed in the following experimental section, which shall beconsidered as illustrative only, and not limiting the scope of thisapplication.

EXAMPLES

Material and Methods

Animals.

Pregnant and adult (six to eight week old, female) C57Bl/6 mice werepurchased from Charles River Laboratories (Les Oncins, France). Neonateswere injected on the day of birth (post-natal 1, PN1). SMA cat breeders(heterozygous and affected animals) were obtained from Dr. Fyfe(Laboratory of Comparative Medical Genetics, Michigan, US) and housed inthe Center of Boisbonne at the Nantes Veterinary School. Genotyping ofSMA kittens was performed as described previously (Fyfe, Menotti-Raymondet al. 2006). Experiments were approved by the regional ethic committee(CREEA).

All animal experiments were carried out according to the Europeanguidelines for the human care and use of experimental animals.

Vector Preparation.

Pseudotyped AAV2/1 and AAV2/9 vectors were generated by packagingAAV2_based recombinant single-stranded (ss) and self-complementary (sc)genomes in AAV1 and 9 capsids. Briefly, the vectors were produced usinga helper-virus free three-plasmid transfection in HEK293 cells with (1)the adenovirus helper plasmid (2) the AAV packaging plasmid encoding therep2 and cap1 or 9 genes (pLTRCO2 for AAV1 and p5E18-VD2/9 for AAV9) (3)the AAV2 vector plasmid containing mSeAP or GFP (under control of thecytomegalovirus immediate early (CMV IE) promoter) as ss or sc genome(Xiao, Li et al. 1998). This latter plasmid was constructed by deletingthe D sequence and the terminal resolution site (trs) site from one ofthe inverted terminal repeat. The recombinant vectors were purified bydouble-CsCl ultracentrifugation followed by dialysis againstphosphate-buffered saline. Physical particles were quantified by realtime PCR for vectors injected in mice and by dot blot hybridisation forvectors injected in kittens and the vectors titers were expressed asviral genome per milliliter (vg/ml).

In Vivo Injection of the AAV Vectors

Neonates mice were injected on the day of birth (post natal 1, PN1). Fori.m. injections, AAV vector solutions (ssAAV2/1 (n=2), ssAAV2/9 (n=2),scAAV2/1 (n=2) or scAAV2/9 (n=3) encoding mSeAP or GFP were injectedinto both triceps and gastrocnemius muscles (1 injection site permuscle, 5 μl per injection, 8×10⁺⁹ to 2×10⁺¹⁰ viral genome per mouse).For i.p. injections, the viral solutions (ssAAV2/1, n=2, ssAAV2/9, n=1,scAAV2/1, n=1 and scAAV2/9, n=2) encoding mSeAP or GFP were injectedinto the peritoneal cavity of one day old C57Bl/6 mice (100 μl, 3×10⁺¹⁰to 10⁺¹¹ viral genome per mouse). For i.v. injections, one day oldC57Bl/6 mice were injected into the temporal vein with scAAV2/9-GFPvector (50 μl, 1.5×10+10 viral genome per mouse, n=3). Adult C57Bl/6mice were injected into the tail vein with scAAV2/9-mSeAP orscAAV2/9-GFP vector (500 μl, 3×10⁺¹¹ vg per mouse, n=3).

At 2 days after birth, a total of 1.5×10⁺¹² vector genome-containingparticles of scAAV9-CMV-eGFP were injected into the jugular vein of oneSMA-affected kitten and one SMA-heterozygous kitten.

Perfusion and Tissue Processing for Histology

Muscles, brains and spinal cords were removed at 1 (PN2), 3 (PN4) or 7(PN8) days post-injection from neonate mice or 7 and 35 dayspost-injection from adult mice. Adult C57Bl6 mice were anesthetized(xylazine 10 mg/kg, ketamine 100 mg/kg) and perfused intracardially with0.1 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde(PFA) in PBS. Tissues were removed and post-fixed for 4 h in the samesolution before being transferred overnight at 4° C. into 15% sucrosefor brains and muscles and 30% sucrose for the spinal cords. Neonateswere decapitated and tissues were immersed in 4% PFA for 4 h beforebeing cryoprotected overnight at 4° C. Samples were frozen in coldisopentane (−50° C.) and serial sections were cut in a cryostat andstored at −80° C. for further analysis.

At 10 days post-injection, kittens were anesthetized (medetomidine 150μg/kg, ketamine 10 mg/kg) and perfused transcardially with 10 ml ofphosphate-buffered saline followed by 100 ml of 4% PFA. Brains andspinal cords were removed and cut into coronal 5 mm slabs then postfixedin 4% PFA following by overnight cryoprotection in 30% sucrose and thenfrozen on dry ice in OCT compound. Spinal cord slices were cut inintervals of 1×100 μm followed by 5×10 μm in a cryostat. Hundredμm-thick sections were used for examination of GFP signal by confocalmicroscopy, and 10 μm-thick sections were used for immunocytochemistry.

Evaluation of Transgene Expression

For mSeAP histochemistry, muscles, brains and spinal cords from neonatemice were removed at 1, 3 and 7 days p.i., frozen in cold isopentane(−50° C.), and maintained at −80° C. for extemporal use. Brain andspinal cord from adult animals were harvested at 35 days p.i and treatedunder the same conditions. Tissue sections of 16 μm thick for brain andspinal cord, and 8 μm thick for muscles were performed in a cryostat andsubsequently processed for transgene expression. The sections were fixedwith 0.5% glutaraldehyde, washed with PBS and endogenous alkalinephosphatase was heat-inactivated for 30 min at 65° C. Sections were thenincubated overnight at 37° C. in 0.165 mg/ml5-bromo-4-chloro-3-indolylphosphate and 0.33 mg/ml of nitrobluetetrazolium in 100 mM Tris-HCl, 100 mM NaCl and 50 mM MgCl2,counterstained with hematoxylin-eosin, and mounted with Eukit.

For GFP immunohistochemistry in mice, sections were washed in PBS andincubated for 30 min in a solution of hydrogen peroxide(Peroxydase-Blocking solution, Dako) for inhibition of the endogenousperoxidases. After washing in PBS, sections were blocked for one hour atroom temperature in PBS with 10 goat serum (Dako) and 0.4% Triton andthen incubated overnight with a rabbit polyclonal anti-GFP (Abcam;1:3000). A biotin-conjugated secondary antibody (Vectastain, 1:200) andthe Vectastain Elite ABC kit were used, and DAB staining was revealedwith the DAB substrate kit for peroxydase (Vector Laboratories).Sections were dehydrated in alcohol and xylen, and mounted with Eukit.

GFP immunocytochemistry in cat was realised on 10 μm spinal cord frozensections. Briefly, spinal cord sections were permeabilized with 0.2%Tween 20 in PBS (pH 7.4), blocked with 5% goat serum, incubated twonights at 4° C. with polyclonal antibody AB3080 to GFP (Chemicon, 1:50)and incubated with a biotinylated goat anti-rabbit antibody. Theimmunolabeling was revealed after an incubation with thestreptavidine-peroxydase complex by using the diaminobenzidine substrateperoxydase. The sections were counterstained with haematoxylin.

Motor neuron's choline acetyltransferase was labeled with cholineacetyltransferase (ChAT) goat polyclonal ChAT antibody (AB144P,Chemicon, France, 1:100). Briefly, spinal cord sections were blockedwith 1% rabbit serum in PBS/Tx100 0.4%, incubated one night at roomtemperature with the primary antibody and incubated with a biotinylatedrabbit anti-goat antibody. The immunolabeling was revealed after anincubation with streptavidine alexa 555 fluor and sections werecoverslipped with Mowiol medium (Calbiochem, USA) to be viewed underconfocal microscopy.

Laser Confocal Scanning Microscopy

GFP expression and immunocytochemistry were observed with an invertedNikon TE-2000 laser scanning confocal microscope, equipped with a blueargon ion laser and a helium neon laser emitting monochromatic rays at488 nm (green) and 543 nm (red), respectively. Slides were scannedserially using a water immersed X20 objective. Each image was recordedin a separated channel (channel green for GFP and channel red forstreptavidin 555) and overlayed to allow detection of colocalizedfluorescent signals.

Example 1. Intramuscular Injection of mSEAP-Expressing AAV Vectors inthe Neonatal Mouse

We first evaluated the potential of serotype 1 and 9 ss- or scAAVvectors to transduce the CNS cells after i.m. injection. The ssAAV1,ssAAV9, scAAV1 or scAAV9 encoding the murine secreted alkalinephosphatase (mSEAP) under the cytomegalovirus (CMV) promoter wereinjected into both triceps and gastrocnemius muscles in one day oldC571316 mice (8.10+9 to 2.10+10 viral genome per mouse, 3 mice pergroup). The injected muscle, brain and spinal cord tissues were removed1, 3 or 7 days post injection and analysed for mSEAP expression usinghistochemistry.

The mSEAP expression was detected in the injected muscles 3 and 7 daysafter injection of each AAV serotype, except with ssAAV9, the expressinglevel dramatically increasing with time (FIG. 1 a ). In the CNS,transgene expression was detected only after i.m. injection of scAAV9.Interestingly, the mSEAP expression was detected in the epithelial cellsof the choroids plexus (FIG. 1 b ), which have a crucial role in thesecretion and the clearance of many proteins and toxins in thecerebrospinal fluid (CSF) (Redzic, 2005). The mSEAP expression in thechoroids plexus was found as soon as 3 days post-injection (PN4), andthe expression levels again increased with time. A weak transgeneexpression was also located in and around blood vessels of the brain andthe spinal cord after i.m. injection of the scAAV9 vector (FIG. 1 c ).

Example 2. Intraperitoneal Injection of mSEAP-Expressing AAV Vectors inthe Neonatal Mouse

We then analysed whether i.p. administration of ssAAV1, ssAAV9, scAAV1and scAAV9 in one day old C57Bl6 mice (100 μl, 3.10⁺¹⁰ to 1.10⁺¹¹ viralgenome per mouse) could mediate transgene expression in the CNS at 1, 3or 7 days post injection.

A low level of mSEAP expression was detected in the diaphragm musclefibres of mice injected with ssAAV1 by 3 days post-injection, which wassimilar to that observed with scAAV1 (FIG. 2 a ), and with both vectorsat 7 days after injection. (FIG. 2 a ). SsAAV9 transduce a few musclefibres only at 21 days post injection, whereas an intense mSEAP stainingwas found in the diaphragm when using scAAV9 from 3 days post-injection(FIG. 2 a ). This high level of transduction was also observed in othermuscles such as the triceps brachii or gastrocnemius muscle (data notshown).

The epithelial cells of the choroids plexus and the ependyma appearedclearly labelled after injection with scAAV9 (FIG. 2 b ). A robusttransduction was further observed in these regions at 7 dayspost-injection (FIG. 2 b ). Transgene expression was also observedwithin the meninges and blood vessels at 7 days post-injection, both inthe brain and throughout the spinal cord, and was higher than thatobserved after i.m. injection (FIG. 2 c ). Interestingly, mSEAPexpression was also detected in some neural cells in the brain and thespinal cord (FIG. 2 d ). Taken together, these results indicate thati.m. or i.p. injected scAAV9 vectors that express the mSEAP protein canefficiently target the CNS, especially the epithelial cells of thechoroids plexus and the ependyma.

Example 3. Transgene Expression in the CNS after i.m. Or i.p. Injectionof scAAV9-GFP in the Neonatal Mouse

Since mSEAP is a secreted protein, the transgene expression observed inthe CNS cells after peripheral AAV injection could result from proteintranscytosis rather than from AAV cell transduction. We thus verifiedwhether similar results could be obtained when using a non-secretedprotein.

A recombinant scAAV9 vector expressing the “green fluorescent protein”(GFP) was injected in neonatal mice either intraperitoneally (3.10+10 vgper mouse, 100 μl) or intramuscularly (8.10+9 vg in 20 μl per mouse, 5μl per muscle). Seven days later, and similarly to that observed withscAAV9-mSEAP, the GFP expression was observed in the choroids plexus andependyma cells located in the brain ventricles (FIG. 3 a and FIG. 4 a ).Furthermore, we found in this case many GFP-positive neural cells inseveral brain regions located, in particular, close to the ventricles.Cell bodies and fibres in the hippocampus (FIG. 3 b ), the septum (FIG.4 b,c ) and the entorhinal cortex (FIG. 3 c , FIG. 4 d-e ) appearedefficiently transduced.

Importantly, GFP expression was detected in cells of the spinal cord at7 days after AAV administration, including in cells with a motorneuron-like phenotype (FIG. 3 d,e ). A strong GFP expression was alsofound in fibres of the corticospinal tract that cross at the cervicalspinal cord level (FIGS. 3 f and 4 h ). Transduction of these fibreslikely results from targeting of the upper MNs whose somas are locatedin the motor cortex and which also appeared GFP-immunopositive (FIG. 3 d). Globally, a higher number of GFP-immunopositive cells were detectedin the CNS after i.p. than after i.m. injection, due to either thedifference of efficacy between the routes of injection or to the highertitre of vector used in the i.p. procedure.

Example 4. Transgene Expression in the CNS after Intravenous Injectionof GFP-Expressing scAAV9 in the Neonatal Mouse

Since recombinant scAAV9 vector appeared as the most efficient one formediating CNS cell transduction after i.m or i.p delivery, we evaluatedwhether this could be improved by using the i.v. route ofadministration.

GFP-expressing scAAV9 vectors were thus injected into the temporal veinof one day old C57Bl6 mice (50 μl, 1.5·10⁺¹⁰ viral genome per mouse) andthe CNS tissues were removed and processed for immunostaining 7 daysthereafter. A strong GFP expression was detected in both the choroidsplexus and ependyma cells (FIG. 5 a ) and in the brain blood vessels(FIG. 5 c ). Again, we found GFP expression within cells of bothneuron-like and glial-like phenotype throughout the brain, in particularin the entorhinal cortex (FIG. 5 d ) and the hippocampus (FIG. 5 e,f ).

A very high level of transgene expression was found throughout thespinal cord (from the cervical to the lumbar segments) in cells with amotor neuron-like phenotype and location (ventral spinal cord) (FIG. 5h-i ). This probably results from diffusion of the vector through theblood vessels from the circulation to the brain parenchyma, or/and toaxonal anterograde transport from upper CNS regions.

We then determine whether ssAAV9 could also cross the BBB and transducethe CNS cells after i.v. delivery, or if this property is specific tothe double-stranded genome. In this aim, GFP-expressing ssAAV9 vectorswere injected into the temporal vein of neonatal mice and GFP expressionwas analyzed 3 weeks after (in order to permit genome conversion intodouble-stranded DNA).

Similar to that observed with scAAV9, ssAAV9-GFP proved to mediate CNScell transduction after i.v. delivery, although its efficacy was lowerthan that of scAAV9. Again, the choroids plexus and ependyma cellsexpressed large amounts of GFP and many brain regions close to thecerebral ventricles were found to be transduced (FIG. 6 a ). Forexample, GFP-positive neurons were detected in the hippocampus and thehabenular nuclei (FIG. 6 a ) and in the median eminence (FIG. 6 b ).Interestingly, some motor neuron-like cells were found to express GFP inthe ventral spinal cord (FIG. 6 c-e ). A few CNS cells were also foundto express GFP after i.m. or i.p. delivery of the recombinant ssAAV9(data not shown).

Altogether, these data suggest an unexpected ability of serotype 9 AAVvectors—either conventional or self-complementary—to cross the BBB andtransduce the CNS cells in neonatal mice, including the lower motorneurons, after a single intravenous injection in the neonatal mouse

Example 5. Intravenous Injection of Ss and scAAV9 Vectors in the AdultMouse

Since the BBB is incompletely formed in neonatal mice, we evaluatedwhether the ability of the AAV9 vectors to transduce neural cells innewborn mice was preserved in adult mice. Ss and sc AAV9 vectorsencoding for mSEAP (3.10¹¹ vg or 1.10¹² vg per mouse) were injected intothe tail vein of adult mice and transgene expression in the CNS wasanalyzed four weeks thereafter. After i.v. delivery of scAAV9-mSEAP, asustained expression of the transgene was found in many brain regionssuch as the median eminence (FIG. 7 f ), the hippocampus (FIG. 7 g ) orthe corpus callosum (FIG. 7 h ).

Importantly, there were many mSEAP-positive cells and fibers throughoutthe spinal cord after i.v. delivery of recombinant serotype 9 AAVvectors (FIG. 8 a-g ). Again, a higher level of transgene expression wasobserved with the sc—than with the conventional ssAAV9 vector (FIG. 8a,b versus 8 c-g).

Similar injections using scAAV9-GFP demonstrated the superiority ofscAAV9 for systemic gene delivery to the spinal cord. A high number ofwas found to express four weeks after i.v. injection of 2×10¹² vgscAAV9, GFP was expressed in the spinal cord in both neuronal and glialcells, as demonstrated using immunostaining of the glial fibrillaryacidic protein (GFAP), a marker of astrocytes (FIG. 11 ).

Hence, our results show an efficient transduction of the CNS cells,including lower MNs and glial cells, after intravenous delivery ofrecombinant AAV9 vectors in adult mice in which the BBB is completelyformed. This emphasizes the particular property of these vectors to passfrom the circulation to the CNS parenchyma through the BBB, achievingwidespread gene transfer to the nervous cells.

Example 6. Intravenous Injection of AAV9-GFP in a Large Animal Model

A validation of this new CNS gene transfer strategy in large animalmodels is a prerequisite to a potential application in human clinics.

We evaluated transgene expression in the spinal cord of LIX-1 kittensfollowing intravenous delivery of recombinant scAAV9 vectors. Two daysold kitten (one LIX-1 homozygous and one heterozygous) were injectedinto the jugular vein with GFP-expressing scAAV9. Ten days after, spinalcord tissue sections were analyzed for GFP expression using laserscanning confocal microscopy.

A strong GFP signal was observed along the spinal cord from the cervicalpart to the cauda equina both in the gray and white matter, theexpression pattern appearing similar in both the heterozygous andaffected animals. Nerve fibers of the fasciculi gracilis and cuneatusdorsal sensory tracts expressed high levels of GFP (FIG. 9 a ).Moreover, GFP expression was detected in a number of cell bodies in theventral spinal cord, after both observation of GFP fluorescence (FIG. 9a,c ) and immunohistochemical analysis (FIG. 9 b-d ). Adouble-immunostaining analysis using antibodies against GFP and cholineacetyl transferase (ChAT) showed that, in both SMA-affected andnon-affected kitten, a significant part of the GFP-positive cells weremotor neurons (FIG. 10 ).

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The invention claimed is:
 1. A method for delivering a transgene acrossthe blood brain barrier to spinal cord motor neurons or glial cells of ahuman subject, and expressing said transgene, said method comprisingintravenously administering, in the absence of a blood-brain barrierdisrupting agent, to the subject from 10¹³ to 10¹⁷ viral genomes of arecombinant double stranded self-complementary human serotype 9adeno-associated virus (scAAV) vector comprising an AAV9-derived capsidand a genome comprising the transgene, such that the scAAV vectorcrosses the blood brain barrier and spinal cord motor neuron or glialcells of said subject are transduced and express said transgene.
 2. Themethod according to claim 1, wherein said genome comprises AAV2-derivedinverted terminal repeats.
 3. The method according to claim 1, whereinthe scAAV vector is administered in an amount comprised between 10¹³ and10¹⁶ viral genomes.
 4. The method according to claim 2, wherein thescAAV vector is administered in an amount comprised between 10¹³ and10¹⁶ viral genomes.
 5. The method according to claim 3, wherein thescAAV vector is administered in an amount comprised between 10¹⁴ and10¹⁶ viral genomes.
 6. The method according to claim 4, wherein thescAAV vector is administered in an amount comprised between 10¹⁴ and10¹⁶ viral genomes.
 7. The method of claim 1, wherein said human subjectsuffers from a disorder of the central nervous system selected from thegroup consisting of spinal muscular atrophy and amyotrophic lateralsclerosis.
 8. The method of claim 1, wherein the vector is a pseudotypedAAV vector.
 9. The method of claim 1, wherein the vector comprises areplication defective AAV genome lacking functional Rep and Cap codingviral sequences.
 10. The method of claim 1, wherein the transgeneencodes a protein selected from the group consisting of growth factors,cytokines, hormones, neurotransmitters, enzymes, anti-apoptotic factors,and angiogenic factors.
 11. The method of claim 1, wherein the vector isa pseudotyped AAV vector comprising an AAV2-derived genome packaged inan AAV9-derived capsid.
 12. The method of claim 1, wherein expression ofthe transgene in the vector is controlled by a ubiquitous, regulated ortissue-specific promoter.
 13. The method of claim 1, wherein thetransgene encodes the survival of motor neuron (SMN) protein.